FIELD OF THE INVENTION
The present invention relates generally to the use of novel fermentation and chromatographic procedures separately and jointly for the production of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa (AVDESP™), in biologically active form from fluids, especially mammalian host cell culture supematants.
BACKGROUND AND PRIOR ART OF THE INVENTION
In 1986, FDA approved human tissue plasminogen activator (tPA; Genentech, CA, USA) protein from mammalian cells to be used for therapeutic purpose. It was the beginning, currently there are many more monoclonal antibodies, which got the regulatory approval. Moreover, several hundreds are in pipeline. Like tPA, most of these proteins are expressed immortalized Chinese hamster ovary (CHO) cells, but other cell lines, such as mouse myeloma (NSO), baby hamster kidney (BHK), human embryo kidney (HEK-293) are approved for recombinant protein production. There are two critical issues during the production of therapeutics (a) time taken to provide the material (b) lowering the price of the material to the common user. Therefore, industry continues to look at new technologies and process development strategies that will reduce timelines and also will help in reducing the cost.
As mentioned, mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post-translational modifications. A variety of mammalian cell expression systems are now available for expression of proteins. Generally expression vectors use a strong viral or cellular promoter/enhancer to drive the expression of recombinant gene. However, the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
In past, various media and methods were used for the cell culture manufacturing of recombinant glycoprotein or monoclonal antibody. Commonly employed bioreactor process includes; batch, semi fed-batch, fed-batch, perfusion and continuous fermentation. The ever-increasing demand of monoclonal antibody and other recombinant proteins in properly glycosyalted forms have increased the prospects of cell culture process development. In addition the regulatory hurdles

imposed on the serum containing process has led to the development of cell culture process in a completely chemically defined environment.
Numerous techniques have in the past been applied in preparative separations of biochemically significant materials. Commonly employed preparative separatory techniques include: ultrafiltration, column electrofocusing, flatbed electrofocusing, gel filtration, electrophoresis, isotachophoresis and various forms of chromatography. Among the commonly employed chromatoghraphic techniques are ion exchange and adsorption chromatography. The extensive application of recombinant methodologies to large-scale purification and production of eukaryotic protein has increased the prospect of obtaining the molecule in required quantity using simplified purification procedures.
Anemia is often an associated condition in patients with chronic kidney disease (CKD). There is a direct relationship between the severity of the anemia and the decline in renal function. This anemia is a source of significant morbidity causing symptoms such as lack of energy, breathlessness, dizziness, angina, and poor appetite and decreased exercise tolerance. The main cause of this anemia is a decreased production of erythropoietin, a naturally occurring hormone mainly produced by the kidney. Much of the impaired quality of life and morbidity suffered by patients with CKD may be a consequence of this anemia and it may have a major impact on their sense of well-being as well as impairing their ability to work and affecting their social and sexual lives. In the past, iron and folate were the main treatments for this condition and blood transfusions, with their associated risks of transmission of infection and induction of cytotoxic antibodies, which could jeopardize a future renal transplant, were used sparingly. In 1983 the cloning of the human gene for erythropoietin was achieved, production of recombinant human erythropoietin (r-HuEPO) followed and by 1986 the efficacy of r-HuEPO treatment in dialysis patients was first demonstrated.
Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects. The aim of inventing this new molecular analogue of recombinant human erythropoietin (r-HuEPO) is to obtain a therapeutic with a longer biological half-life compared to r-HuEPO, allowing a reduction of the frequency of injections necessary to maintain a desired level of systemic haemoglobin and haematocrit. The chronic nature of CRF (unless a subject receives a kidney transplant) means that

treatment may continue for a long part of the subject's life and multiple weekly injections of r-HuEPO can have a major impact on subjects and care givers.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to obtain an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Another main objective of the present invention is to obtain an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) used for production of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin.
Yet another objective of the present invention is to develop a method for construction of an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Still another objective of the present invention is to obtain a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Still another objective of the present invention is to obtain novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
The present invention relates generally to the use of novel chromatographic procedures separately and jointly for the production of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, in biologically active form from fluids, especially mammalian host cell culture supematants.
SUMMARY OF THE INVENTION
Accordingly, the present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR); a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR); novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).

The present invention relates to the use of novel fermentation process for the overexpression of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa in CHO cells.
Summary of the present invention also includes use of novel chromatographic procedures for rapid and efficient recovery of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa from cell culture supernatant.
DESCRIPTION OF FIGURES
Figure 1: Restriction digestion of the vector backbone.
Figure 2: Nutrient consumption and lactate accumulation profile during fermentation run
Figure 3: Cell growth and viability profile during fermentation run
Figure 4: Expression profile of protein during fermentation run
Figure 5: Process chromatogram after HIC
Figure 6: Process chromatogram after Anion Exchange chromatography
Figure 7: Process chromatogram after Weak Cation Exchange chromatography
Figure 8: Process chromatogram after Strong Cation Exchange chromatography
Figure 9: Western Blot Analysis of Drug substance showing comparable immuno-specificity
between RMP and drug substance where Lane No. 1: Molecular weight Marker, Lane No. 2:
RMP: Reference Medicinal Product -Aranesp ™ and Lane No. 3: Formulated Drug Substance.
Figure 10: Comparable hydrophobicity profile between Drug substance and RMP as depicted by
Reverse Phase HPLC profile
Figure 11: MALDI-TOF analysis of the intact molecule has determined the molecular mass of
RMP and Drug substance to be ~37kDa
Figure 12: HPLC-based tryptic peptide mapping analysis.
Figure 13: The in vitro effect of Darbepoetin alfa has been extensively demonstrated using the
reporter cell line TFl Erythroleukamic cell assay. The potency value of the sample is calculated
using CFR 21/part 11 compliance with Parallel line assay (PLA) software, has shown identical
profiles between RMP & Drug substance.
Figure 14: In Vivo Efficacy Study of Recombinant Darbepoetin Alfa for erythropoiesis
stimulation in CDl Female Mouse.
Figure 15: In Vivo Efficacy Study of Recombinant Darbepoetin Alfa for erythropoiesis
stimulation in CDl Male Mouse

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improved process for the cell culture manufacturing of Recombinant Darbepoetin alfa. In particular, the invention provides system that helps in achieving proper glycosylation of Recombinant Darbepoetin alfa. In addition, the invention also helps in maintaining higher cell viability for a longer period of time. The cell culture manufacturing process starts with seeding the bioreactor at a predefined cell density in chemically defined medium. The culture is fed in two stages, primary feeding which is designed to achieve the desired cell growth and, secondary feeding which is designed to maintain the higher cell viability and hyper glycosylation of the Recombinant Darbepoetin alfa. Furthermore the invention also relates to bioreactor operation procedure for the manufacturing of Recombinant Darbepoetin alfa.
This present invention relates to the rapid and efficient recovery of Recombinant Darbepoetin alfa from cell culture supernatant from Cell culture fluid by means of Hydrophobic interaction and Ion exchange chromatography. These chromatographic steps are used for capture of recombinant Darbepoetin alfa. This separation involves in selective binding of the desired compound to specific Hydrophobic interaction resin and then elution with elution buffer. Culture supematants are preferably clarified before chromatographic treatment. Darbepoetin alfa isoforms containing eluent fractions are enriched with biologically active material, but they will be subjected to diafiltration and further processing by Anion exchange chromatographic steps. In this process the active materials are colleted in the elution and enrich the isoforms containing more sialic acid residues. The invention relates to the next chromatographic procedure where the elution from the previous step is diafiltered and fiirther subjected to a weak cation exchange chromatographic step. These processes are used for removal of process related impurities like host cell protein and host cell DNA. The active material is collected in the flow through. The invention also relates to the next chromatographic procedure where the flowthrough from the previous step is diafiltered and ftirther subjected to a strong cation exchange chromatographic step. The active material is collected in the elution The present invention also relates to the recombinant Darbepoetin alfa recovery procedure involving serial application different chromatographic techniques as mentioned previously. All different steps, conditions and compositions are disclosed in the invention.

Example 1:
The nucleotide sequence was codon optimized for efficient and stable expression in Cricetulus griseus (CHO) cell line(SEQUENCE ID 1). The optimization was successfiil as no negative cis-acting sites (such as splice sites, poly(A) signals, etc) that may negatively influence expression were present. GC-content was increased to prolong mRNA half-life. Codon usage was adapted to the bias of Cricetulus griseus (CHO) resulting in a high codon adaptation index (CAI) value. The gene was cloned into expression vector. The expression vector contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial ColEl origin of replication. The terminator region has a SV40 enhancer positioned downstream the SV40 polyadenylation signal. A human gastrin terminator was inserted between the SV40 polyA signal and the SV40 enhancer. Each vector also included two human S/MAR (Scaffold/Matrix Attachment Regions) elements flanking the expression cassette. Integrity of the inserted gene of Darbepoetin alfa in the vector backbone was further checked by restriction digestion analysis. The expected fragments of 3 and 9.5 kbs were observed after digestion of expression vector with PstI and Bglll restriction enzymes. Observed restriction analysis has been depicted in Fig. 1. Stable expression of Darbepoetin alfa was performed using an anchorage independent Chinese Hamster Ovary-Kl-S (CHO-Kl-S) cell line that is approved for industrial applications. The vector was linearized and electroporated into the cell line. After selection with Neomycin, stable cell line was generated.
Example 2:
Before seeding, the Bioreactor was assembled and sterilized by autoclaving at 121°C for 45
minutes. After sterilization, Bioreactor was charged with 7200ml of commercially available
animal component free, chemically defined media. Afterwards, the bioreactor was kept under
positive pressure with air at a flow rate of 0.2 Litre per minute. The bioreactor was aerated over
night for 100% air saturation. The d02 electrode was calibrated after stabilization of dissolved
oxygen value. Sterile connection was created between the seed bottle and the seed port on the
bioreactor head plate. The seed was then aseptically transferred to the bioreactor using peristaltic
pump. The bioreactor was seeded with the density of 0.4 x 10* cells/mL. After seeding, the
bioreactor was allowed to run at following pre-set parameters:
pH: 6.9-7.2
d02: 30 - 60% of air saturation Temperature: 30 - 38°C. Stir speed: 70-80RPM

The bioreactor was sampled at every 24/48 hours for in process quality control analysis. The
bioreactor process was a fed - batch process with feeding of different nutrients at definite culture
stages. During first 72 hrs of culture time, the bioreactor was daily fed with 300mL of primary
feed that comprise of glucose, galactose, mannose, lipids, amino acids, vitamins, trace elements,
cholesterol and growth factors. Starting from 96 hrs of culture age the bioreactor was daily fed
with 150mL of secondary feed that comprise of Galactose, trace Elements, Manganese Chloride
and Mannose. During first 120 hrs of culture age the bioreactor was operated at following pre-set
and controlled parameters;
pH: 7.1 ±0.1
d02: 30 - 60% of air saturation Temperature: 36 - 37°C. Stir speed: 70-80RPM
From 120 to 288 hrs the bioreactor was operated at following parameters;
pH: 6.8 ±0.05
d02: 30 - 60% of air saturation Temperature: 32 ± 0.5 °C. Stir speed: 70-80RPM
The bioreactor was harvested at a cell viability of 70 - 80%. The growth pattern, protein expression profile and nutrient consumptions are depicted (Fig 2-4).
Examples:
Clarification of the cell culture harvest was carried out by using a cellulose disposable filter with 650 - 1000 cm^ effective filtration area and with an operating pressure of not more than 30 psi. The filtrate was checked for turbidity and target protein content. The clarified harvest was diafilitered and buffer exchanged with Acetate buffer pH 4.5 - 5.5 using a 30 kDa - TFF membrane at a Trans Membrane Pressure of 5-10 psi. 2 Molar NaCl was added to the diafiltered product. Hydrophobic interaction chromatography was used in binding and elution mode with column of 100 mm diameter for capturing; with Acetate buffer pH 4.5 - 5.5 containing 2 Molar NaCl as equilibration buffer. After the sample is loaded on to the column, it is washed with equilibration buffer. The protein of interest was eluted with Acetate buffer pH 4.5 -5.5 (Fig 5). The HIC elute fraction was buffer exchanged with Tris buffer pH 6.8 - 7.2 using a 30 kDa - TFF membrane at a Trans Membrane Pressure of 5-10 psi. Anion exchange chromatography in binding and elution mode was carried out with column at an operational flow

rate of 10 ml/min. The column was equilibrated with Tris buffer pH 6.8 - 7.2. Protein of interest is collected in the elution. This step was used for the enrichment of specific isoforms of Recombinant Darbepoetin alfa (Fig 6). Thereafter, the elution fraction was diafiltered and buffer exchanged with Tris buffer pH 6.8-7.2. Weak cation exchange chromatography was carried out after equilibrating the column with Tris buffer pH 6.8-7.2. The Protein of interest was in the flow through. This step was used for the removal of process related impurities like host cell DNA and host cell protein (Fig 7). The eluate was buffer exchanged using a 30 kDa TFF membrane at a Trans Membrane Pressure (TMP) of 5-10 psi using Acetate buffer pH 3.6-4.5. Strong Cation exchange chromatography was carried out on buffer exchanged protein solution in binding and elution mode using Acetate equilibration buffer pH 3.6-4.5. Protein of interest was collected in the elution (Fig 8). Thereafter the elution fraction was filtered for virus removal using viral removal filter having an effective filtration area of 0.001 m^ The nano filtrate was buffer exchanged with Phosphate buffered saline pH 6.2±0.2 to get the drug substance, which was finally filtered using 0.2|am filter. The drug substance was characterized as per the specifications. The Drug Substance (Active Pharmaceutical Ingredient) was formulated by adding Polysorbate 80 to a final concentration of 0.05 mg/ml of protein solution to get the drug product.
Example 3:
The formulated material was characterized as per the specifications set by product development specification. Western blot analysis for this protein showed a single diffused band corresponding to 48-50 Kda aligning with the RMP (Fig 9). The isoelectric focusing showed a pi value approximately 3.0 aligning with RMP. This drug substance when analysed in Reverse Phase HPLC showed the retention time of 15.222 minutes with the test sample in comparison to RMP value of 15.350 minutes(Fig 10). Size Exclusion HPLC also revealed retention time of 8.800 minutes in comparison to the retention time 8.767 of RMP. Intact molecular mass estimation performed by high-sensitivity MALDI-TOF MS analysis has revealed the molecular mass of purified Darbepoetin alfa and RMP to be 37.5 kDa (Fig 11). The results obtained Peptide Mapping by HPLC showed a similar and corresponding profile to RMP(Fig 12). Glycan profiling and Deglycosylation analysis using N-Glycanase has revealed a comparable glycosylation profile between the purified product tested and RMP. The N-terminal analysis showed a corresponding sequence with the RMP. The Relative potency analysis by In vitro TFl Erythroleukemic Cell based bioassay showed 1.05 in comparison to RMP (Fig 13).

Example 4:
Acute Intravenous Toxicity and Subcutaneous Toxicity Study of Darbepoetin alfa in Wistar Rat were carried out with a human equivalent dose of lOX. The same study of intravenous and subcutaneous was also carried out in the similar way in Swiss Albino mice. From the studies, inference drawn were that no clinical signs of toxicity was observed at single dose administration of Darbepoetin Alfa by intravenous and subcutaneous routes in rat and mouse. In the next study, In Vivo Efficacy Study of Darbepoetin Alfa in CDl mice for erythropoiesis stimulation was carried out. Statistically significant changes in RBC count, Haemoglobin level, Haematocrit percentage were observed in the group's animal treated with biosimilar Darbepoetin Alfa, Aranesp and Epocrit (Fig 14-15). From the study it had been inferred that a six times lower dose of Darbepoetin alfa has shown equivalence effect with recombinant Human erythropoietin. Repeated dose 28 - day Intravenous toxicity study of biosimilar Darbepoetin Alfa in Wistar rats and New Zealand White rabbits were also carried out to evaluate the toxicological profiles of Darbepoetin alfa in comparison to the commercially available drug. The study data infers that the test substance did not produce any significant adverse effect even at high dose i.e. 15.5 mcg/kg for rabbit and 31 mcg/kg for rat. A Repeated Dose Developmental toxicity study of biosimilar Darbepoetin Alfa in pregnant Wistar Rats was also carried out. Based on the findings, inference was that the test substance did not produce any significant adverse effect on repeated dose during gestation period to the dams as well as the pups. Further study on skin sensitization potential also inferred that Darbepoetin alfa is a non-sensitizer.

We claim:
1. An isolated nucleic acid comprising one or more sequences selected from the group
consisting of SEQ ID NO: 1, complements, variants, and functional fragments thereof and sequences being at least 70% homologous thereto.
2. The isolated nucleic acid of claim 1, wherein the biomolecule is a protein.
3. The isolated nucleic acid of claim 1, wherein the biomolecule is a Darbepoietin alfa
4. A method for constructing an expression vector having increased expression efficiency, the method comprising inserting the isolated nucleic acid of claim 1 into an expression vector.
5. The method according to claim 4, wherein the expression vector is a mammalian expression vector.
6. A vector comprising the isolated nucleic acid of claim 1.
7. The vector of claim 6, wherein said vector is a bacterial plasmid, a bacteriophage vector, a yeast episomal vector, an artificial chromosomal vector, or a viral vector.
8. The vector of claim 6, wherein said vector is a mammalian expression vector.
9. A method for producing a recombinant host cell, the method comprising introducing the isolated nucleic acid of claim 1 or the expression vector of claim 6 into a host cell.
10. The method according to claim 6, wherein the isolated nucleic acid or the expression vector is introduced by way of transfection.
11. The method according to claim 6, wherein the isolated nucleic acid gets integrated with the genome of the recombinant host cell upon transfection.
12. A host cell produced according to the method of claim 11.
13. A host cell comprising the vector of claim 11
14. The host cell according to claims 12 or 13, wherein said host cell is a eukaryotic cell.
15. The host cell according to claims 12 or 13, wherein said host cell is a mammalian cell.
16. The expression vector of claim 4, wherein the protein is recombinant Darbepoietin alfa.
17. The host cell according to claims 12 or 13, wherein the protein produced is recombinant Darbepoietin alfa.
18. A process for recovering Recombinant Darbepoetin alfa comprising steps of:

a) contacting culture supematant(s) with resin(s) for selective adsorption of compound(s) through hydrophobic interactions;
b) eluting the adsorbed compound with eluant followed by enriching with biologically

active material; and c) subjecting the enriched product to anion exchange chromatography to obtain the
Recombinant Darbepoetin alfa. d)subjecting the enriched product to cation exchange chromatography in flow through
mode to obtain the Recombinant Darbepoetin alfa
e) subjecting the enriched product to cation exchange chromatography in binding and
elution mode to obtain the Recombinant Darbepoetin alfa
f) subjecting the enriched product to combination of Anion and Cation exchange
chromatography to obtain the Recombinant Darbepoetin alfa.
19. The process as claimed in claim 18, wherein said supernatant is mammalian host cell culture supernatant.
20. The process as claimed in claim 18, wherein said supernatant is cell culture derived fluid.
21. The process as claimed in claim 18, wherein said supernatant is a mammalian cell culture derived fluid.
22. The process as claimed in claim 18, wherein said culture supematant(s) are concentrated and clarified before contacting resins.
23. The process as claimed in claim 18, wherein the process removes host cell protein and host cell DNA from culture supernatant.
24. A cell culture manufacturing process for the manufacturing of recombinant Darbepoetin.
25. pH of the Acetate buffer is between 4.5-5.5 in hydrophobic interaction.
26. A specific low pH (2-4) wash step during strong anion exchange chromatography for enrichment of high sialiated isoforms.
27. A method of producing Darbepoetin in a large-scale production cell culture comprising mammalian cells that contain a gene encoding Darbepoetin
28. A process of increasing protein glycosylation using a predefined secondary feed.
29. The process as claimed in claim 27, where secondary feed can be comprised of Carbohydrates.
30. The process as claimed in claim 27, where secondary feed can be comprised of Carbohydrates and Trace Elements.
31. The process as claimed in claim 27, where secondary feed can be comprised of Carbohydrates, Trace Elements and Manganese Chloride.
32. The process as claimed in claim 27, where secondary feed can be comprised of Galactose and Mannose

33. The process as claimed in claim 27, where secondary feed can be comprised of 1-2 Molar
Galactose and Mannose
34. The process as claimed in claim 27, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and Mannose
35. The process as claimed in claim 27, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and Mannose along with Trace Elements
36. The process as claimed in claim 27, where secondary feed can be comprised of 25-75%
of 1-2 Molar Galactose and Mannose along with 0.01 - 1.0% Trace Elements and
Manganese Chloride.
37. The process as claimed in claim 27, where secondary feed can be comprised of 25-75%
of 1-2 Molar Galactose and Mannose along with 0.01 - 1.0% Trace Elements and 0.02 -
5 micro molar Manganese Chloride.